The therapeutic utilization of peptide-based pharmaceuticals for many disorders has been the focus of numerous studies. However, the therapeutic utilization of peptide-based drugs has been somewhat confounded because of the poor bioavailablity due to metabolic and chemical degradation, and that they tend to be very polar molecules that do not readily traverse biological barriers. The key to successfully oral dosage form design for these compounds is to put into perspective the biochemical and physical barriers that the dosage form must overcome for effective drug delivery. One way to potentially overcome these barriers is to design the peptide-based drugs with molecular functional recognition characteristics for facilitative nutrient transporters, i.e., proton/oligopeptide (POT) transporters, and then targeting dosage form drug release in regions of high transporter expression. In order to achieve this, one must first identify the transporter(s) responsible for peptide-based drug transport, and their activity and expression patterns in the GI tract. The main objective of this proposal is to initiate the investigation of these three critical factors in the human GI tract. Peptide Transporter 1 (PepT 1), has been traditionally believed to be the predominant GI POT member. However, its apical localization does not provide a means for the cellular exit of the drug at the basolateral membrane. Studies from our laboratory, as well as others, suggest that the recently identified POT members, peptide/histidine transporter 1 (PHT1) and peptide/histidine transporter 2 (PHT2), may provide additional insights into how peptides and peptide-based drugs cross the intestinal epithelium. Based on these results we hypothesize that PHT1 and PHT2 in addition to PepT1, play crucial roles in the facilitation of intestinal transepithelial transfer of peptides and peptide-based drugs. We also hypothesize that the regional patterns of expression and functional differences in the activities of these POT transporters may contribute to errors in the overall evaluation of intestinal transport/absorption kinetic assessments of di- and tripeptides and peptide-based drugs where they are not all considered. We plan to investigate the individual contributions to the intestinal transport of peptide and peptide-based drugs through the following specific aims: Specific Aim 1: To characterize the spatial patterns of expression and cellular localization of the PepT1, PHT1, and PHT2 in the distinct regions of the human gastrointestinal tracts and in the HT-29 cell line. Specific Aim 2: Utilizing an epitope-tagging strategy, we will generate stably transfected Mandin-Darby Canine Kidney (MDCK) cells that will enable us to delineate the individual pharmacological function of the human isoforms of PepT1, PHT-1, and PHT2 transporter isoforms. Kinetic differences between the Michaelis-Menten kinetics and transport measurements for a range of peptides and peptide-based compounds in each transporter clone and will be contrasted; and Specific Aim 3: To identify and investigate the mRNA expression of known splice variants of PepT1, PHT1, and PHT2 genes in the human sample GI tracts.. Our proposal represents the first step towards the characterization of novel human peptide transporters and will potentially have a major impact on our current understanding of peptide and peptide-based drug transport in the human GI tract.